In vitro adenoviral vector p53-mediated transduction and killing correlates with expression of coxsackie-adenovirus receptor and alpha(nu)beta5 integrin in SUDHL-1 cells derived from anaplastic large-cell lymphoma.

Adenoviral vector-mediated p53 expression induced apoptosis is a well established gene therapy approach that has been evaluated extensively in epithelial tumors but only recently in lymphoid malignancies mainly due to the known resistance of the lymphoid lineage to adenovirus infection. Recently, it was shown that this resistance is not absolute and that cell lines derived from anaplastic large cell lymphoma (ALCL) and some other lymphoid malignancies are efficiently transduced by adenoviral vectors. Normal circulating T lymphocytes do not express coxsackie-adenovirus receptor (CAR) and alpha(nu)beta integrins and are relatively resistant to infection by adenovirus. These molecules serve as receptors for adenovirus entry into the cells. ALCL-derived SUDHL-1 cells were evaluated for transduction efficiency and expression of p53 after infection with an adenoviral vector containing wild-type p53 (AdWTp53). Cells derived from ALCL and circulating mononucleated cells (MNCs) were also evaluated for expression of CAR and alpha(nu)beta integrins. AdWTp53-mediated expression of p53 resulted in p21/WAF1 induction, G1 arrest, and apoptosis in SUDHL-1 cells. The expression of CAR and alpha(nu)beta5 integrin was high in SUDHL-1 cells and comparable to levels observed with epithelial tumor cells, but it was absent in MNCs. The susceptibility to adenoviral vector transduction of the tumor-derived cells implies an important biological difference between them and circulating MNCs, possibly underlying the malignant transformation that ALCL cells undergo. Further studies will be required to evaluate this initial observation in more cell lines and tissue derived from ALCL.     

Reduced expression of coxsackievirus and adenovirus receptor (CAR) in tumor tissue compared to normal epithelium in head and neck squamous cell carcinoma patients

Even though adenoviral vector is widely used in gene therapy of squamous cell carcinoma of the head and neck (SCCHN), the expression level of Coxsackievirus and adenovirus receptor (CAR) in SCCNH is not clearly defined. To identify this variability, the expression of CAR was measured using SCCHN cell lines and compared with transfection efficiency. It was found by RT-PCR and Western blot analysis that CAR levels varied in SCCHN cell lines. FACS analysis and adenovirus infection assay revealed that there was a good correlation between the level of CAR expression and the transfection efficiency. To identify the actual CAR expression patterns of human SCCHN tissues in vivo, immunohistochemical staining was undertaken on frozen biopsies of six SCCHN patients. In all the patients examined, the normal tissues showed much stronger staining for CAR than the tumor tissues. These results demonstrate that the level of CAR expression of a tumor should be evaluated before clinical application of adenoviral vector for gene therapy in SCCHN.     

Late resistance to adenoviral p53-mediated apoptosis caused by decreased expression of Coxsackie-adenovirus receptors in human lung cancer cells

Adenovirus-mediated wild-type p53 gene transfer induces apoptosis in a variety of human cancer cells. Although clinical trials have demonstrated that a replication-deficient recombinant adenovirus expressing the wild-type p53 gene (Ad-p53) is effective in suppressing growth of non-small cell lung cancer (NSCLC), we often experienced late resistance to this treatment. To elucidate the mechanism of late resistance to Ad-p53 in human lung cancer cells, we generated 5 different resistant variants from p53-susceptible H1299 NSCLC cells by repeated infections with Ad-p53. We first examined the transduction efficiency of adenoviral vector by Ad-LacZ transduction followed by X-gal staining in parental and 5 resistant H1299 cell lines. Their sensitivity to viral infection decreased in correlation with the magnitude of resistance, and Ad-p53-mediated tumor suppression could be restored by dose escalation of Ad-p53 in the resistant variants. The expression of Coxsackie and adenovirus receptor (CAR) and alphaV integrins, which are cellular receptors for attachment and internalization of the virus, respectively, was next investigated in these cell lines. Flow cytometry revealed that alphaVbeta3 and alphaVbeta5 integrin expression was consistent, while p53-resistant cell lines showed that diminished CAR expression correlated with the magnitude of the resistance. Our results demonstrated that decreased CAR expression could be one of the mechanisms of late resistance to Ad-p53, which may have a significant impact on the outcome of adenovirus-based cancer gene therapy.     

Reduced transduction efficiency of adenoviral vectors expressing human p53 gene by repeated transduction into glioma cells in vitro.

PURPOSE: Recombinant adenoviral vectors are widely used in clinical and experimental studies to treat malignant tumors. Recently, host immune responses have been proposed as a major limitation in using adenoviral vectors for repeated gene delivery. We demonstrate another limitation unrelated to host immunity. EXPERIMENTAL DESIGN: We repeatedly transduced an adenoviral vector expressing the human p53 gene (AxCIhp53) into U373MG, a p53-susceptible cell line, and established the AxCIhp53-resistant cell line U373R. Most U373R cells survived even after AxCIhp53 treatment due to reduced transduction efficiency. Expression levels of adenovirus receptors were estimated to investigate the cause of reduced transduction efficiency. The mutant vector was used to overcome the resistance. RESULTS: The transduction efficiency of an adenoviral vector possessing the reporter LacZ gene (AxCAZ2-F/wt) for U373R cells was 25.4-fold less than that for parent cells. The expression levels of integrins alpha(v)beta(3) and alpha(v)beta(5) were found to be decreased in U373R cells without affecting the expression levels of Coxsackievirus and adenovirus receptor. The mutant vector AxCAZ2-F/K20, with a linker and a stretch of 20 lysine residues at the COOH-terminal of the fiber protein, improved the transduction efficiency of U373R cells to 12.6-fold of that of AxCAZ2-F/wt. A mutant vector carrying the p53 gene, AxCAhp53-F/K20, dramatically induced apoptosis in U373R cells. CONCLUSIONS: Glioma cells expressing low levels of adenovirus receptors might survive and proliferate to recur after repeated adenoviral transduction, even if the adenoviral transduction is effective at first. Changing the tropism of vectors is a potent method to overcome resistance.     

Cancer cells as targets for lentivirus-mediated gene transfer and gene therapy

Lentiviruses have been used as gene transfer vectors for almost 10 years and their utility has been demonstrated in a variety of different applications. However, their value in cancer gene therapy has not been studied thoroughly. Here we show that VSV-G pseudotyped HIV-1-based lentiviruses are efficient vectors for human tumor cells in vitro and in vivo. Lentiviral gene transfer efficiency was demonstrated by transducing 42 different cell lines, representing 10 different human tumor types. It was shown that most of the cell lines were good or excellent targets for lentiviral transduction, allowing 50-95% gene transfer efficiency. These results were comparable to those obtained with an E1/E3 deleted, serotype 5 adenovirus vector. Analysis of lentivirus vector structure revealed that virus particles devoid of HIV-1 accessory proteins appeared to be more efficient, but the presence of enhancing elements cPPT and WPRE did not play a major role in transduction efficiency to four different human tumor cell lines. However, their effect on the gene expression level in these cells was apparent. To examine the impact of lentiviral gene expression level on suicide gene therapy approach, human osteosarcoma cells were transduced with lentivirus- or adenovirus vectors carrying the fusion gene HSV-TK-GFP and exposed to ganciclovir. Cell viability analysis after the treatment revealed that both vector types induced similar level of cytotoxicity, suggesting that lentiviral expression of a suicide gene is adequate for tumor cell destruction. Finally, in vivo transduction studies with subcutaneous tumors showed that lentivirus vectors can yield similar gene transfer efficiency than adenovirus vector, despite three orders of magnitude lower titer of the lentiviral preparation. In conclusion, these data show that lentiviruses are efficient gene transfer vehicles for human tumor cells and justify their use in further preclinical cancer gene therapy studies.     

Differential effects of adenovirus-p16 on bladder cancer cell lines can be overcome by the addition of butyrate.

High frequency of p16 alteration and high local recurrence rate of bladder cancer make this cancer an ideal target for p16 gene therapy. However, a low transduction rate of p16 via adenoviral vector causes an inconsistent result. In this study, we have tested adenovirus-p16 in several bladder cancer cell lines and investigated a way of improving the low transduction rate. Adenovirus-p16 showed a strong antitumor effect on bladder cancer cell lines (253J and T24) with strong Coxackie-adenoviral receptor (CAR) expression but little antitumor effect on bladder cancer cell lines (J82 and HT1376) with little CAR expression. In this study, we suggest a simple way of overcoming the differential effects of the adenovirus. The addition of butyrate to media was found to increase the transduction rate of adenovirus remarkably and increase the antitumor effect of adenovirus-p16 in bladder cancer cell lines with little CAR expression. Butyrate effects were related with increased CAR expression on the cell surface as well as increased transgene expression from adenoviral vector. From these observations, application of adenovirus-p16 gene therapy with butyrate can overcome the obstacle of low gene transfer and enhance the antitumor effect of adenovirus-p16 in bladder cancer.     

Histone deacetylase inhibitors upregulate expression of the coxsackie adenovirus receptor (CAR) preferentially in bladder cancer cells

Studies on bladder cancer cell lines have shown that low adenoviral (Ad) infectivity is associated with low-level coxsackie adenovirus receptor (CAR) expression. Recently, we and others demonstrated a tumor stage- and grade-dependent downregulation of CAR expression in a large series of clinical bladder cancer specimens. Here, we demonstrate adenoviral gene transfer can be markedly enhanced in bladder cancer cells by upregulation of CAR through the use of certain differentiating agents, including the histone deacetylase inhibitors (HDACI) trichostatin A and sodium phenylbutyrate. CAR upregulation to supraphysiologic levels was demonstrated by quantitative rt-PCR, Western blotting, flow cytometry and adenoviral gene transfer. Normal urothelial cells and CAR-positive papilloma cells (RT4) failed to demonstrate upregulation under the same conditions. Upregulation was cell cycle dependent, associated with increased adenoviral gene transfer and persisted for at least 7 days after a single treatment. Such upregulation, however, appears to be tumor cell specific, as other CAR-negative cell lines failed to demonstrate enhanced adenoviral gene transfer with the same treatments. These results provide a rational basis for combining HDACI therapy with gene therapy as a method of augmenting activity in bladder cancer, but this strategy may not be universally applicable to other cell types.     

Variability of adenovirus receptor density influences gene transfer efficiency and therapeutic response in head and neck cancer.

Despite encouraging preclinical studies in many tumor types including head and neck squamous cell carcinoma (HNSCC), initial clinical trials with adenovirus-mediated gene therapy have been disappointing. Although the adenovirus is a "highly efficient vector," it is still limited by the extent of effective in vivo transduction. In our studies with multiple human HNSCC cell lines, we have noted a variation in both in vitro and in vivo responses to the same recombinant adenovirus therapeutic construct. We hypothesize that adenovirus receptor density among tumor cell populations, even of the same histology, greatly influences transduction efficiency and therapeutic results of a variety of adenovirus-based gene therapy strategies. To investigate this hypothesis, the numbers of adenovirus receptors on three well-characterized HNSCC cell lines were determined. Marker and cytokine gene transfer efficiencies as well as therapeutic outcomes after adenovirus-mediated tumor suppressor gene and suicide gene therapies were evaluated and correlated with receptor status. A 5-fold variation in adenovirus receptor density was identified among the HNSCC cell lines (P < 0.002, t test). This variation directly correlated with adenovirus type 5 (Ad5)-mediated green fluorescent protein marker gene and Ad5-interleukin 2 cytokine gene transfer efficiency and resulting protein expression in each individual cell line. The receptor density also directly correlated with therapeutic response after Ad5-thymidine kinase or Ad5-p16 gene transfer in each HNSCC line. The role of the adenovirus receptor in gene transfer efficiency was further supported by recombinant Ad5 fiber knob blocking experiments. The marker gene transfer was increasingly blocked by the same concentration of Ad5 recombinant fiber knob in relation to decreasing levels of adenovirus receptor in the HNSCC lines. An Ad5 recombinant construct that carries the shared coxsackie and adenovirus receptor (CAR) was created and used to up-regulate receptors on each cell line. Ad5-CAR infection significantly increased Ad5-beta-Gal gene transfer efficiency and expression (P = 0.0003, Mann-Whitney test). This increased marker gene expression remained consistent with the established pattern of gene transfer efficiency among the HNSCC cell lines. These data confirm the importance of the adenovirus receptor on individual tumor cell lines with respect to investigating novel adenovirus-mediated gene therapy strategies. This work further supports consideration of assaying adenovirus receptor status, even in tumors of the same histology from patients enrolled in gene therapy clinical trials. Adenovirus receptor status may prove valuable for selecting or stratifying patients as well as assessing outcomes among patients within adenovirus-based cancer gene therapy trials.     

肿瘤细胞表面柯萨奇病毒 腺病毒受体与5型腺病毒感染效率的关系

目的〖HT5"SS〗: 探讨不同肿瘤细胞系柯萨奇病毒腺病毒受体（CAR）和整合素的表达水平与5型腺病毒感染效率的关系,为腺病毒基因治疗研究奠定基础。〖HT5W〗方法〖HT5"SS〗: 利用瞬时转染CAR的真核表达质粒提高肿瘤细胞表面CAR的表达,应用抗体封闭细胞表面的CAR和整合素后,通过流式细胞仪和荧光素酶分析法测定腺病毒Ad5CMVEGFP和Ad5CMVLuc对肿瘤细胞的感染效率和基因表达水平的变化。〖HT5W〗结果〖HT5"SS〗： 不同肿瘤细胞表面CAR和整合素的表达水平是不同的,其中SMMC7721和A549细胞CAR的表达量最高,而K562细胞CAR的表达水平最低;荧光显微镜和流式细胞仪检测Ad5CMVEGFP对肿瘤细胞的感染效率,结果显示5型腺病毒对于SMMC7721和A549细胞的感染效率最高,而对于K562细胞则很低;瞬时转染表达CAR的真核质粒可提高多种肿瘤细胞表面CAR的表达,较大幅度提高了5型腺病毒的感染效率。而抗体封闭肿瘤细胞表面的CAR或整合素后,腺病毒感染效率显著下降。〖HT5W〗结论〖HT5"SS〗:肿瘤细胞表面CAR和整合素的表达水平决定了腺病毒对肿瘤细胞的感染效率。     

Enhanced adenovirus transgene expression in malignant cells treated with the histone deacetylase inhibitor FR901228

The presence of coxsackie and adenovirus receptor (CAR) and alpha(v) integrin on cell surfaces is required for efficient adenovirus infection. Treatment of cells with the histone deacetylase inhibitor FR901228 (depsipeptide) increased CAR and alpha(v) integrin RNA levels in six cancer cell lines. Sodium butyrate and trichostatin A, other histone deacetylase inhibitors, caused similar increases. Cells treated with FR901228 prior to infection had a 4-10-fold increase in transgene expression from a beta-galactosidase-expressing adenoviral vector. These studies suggest that FR901228 increases the efficiency of adenoviral transgene expression and may be useful in cancer gene therapy.     

The therapeutic efficacy of adenoviral vectors for cancer gene therapy is limited by a low level of primary adenovirus receptors on tumour cells

Replication-defective adenoviral vectors are currently being employed as gene delivery vehicles for cancer gene therapy. To address the hypothesis that the therapeutic efficacy of adenoviral vectors is restricted by their inability to infect tumour cells expressing low levels of the primary cellular receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR), we have employed a pair of ovarian cancer cell lines differing only in the expression of a primary receptor for Ad5. This novel system thus allowed the direct evaluation of the relationship between the efficacy of an adenoviral vector and the primary receptor levels of the host cancer cell, without the confounding influence of other variable cellular factors. We demonstrate that a deficiency of the primary cellular receptor on the tumour cells restricts the efficacy of adenoviral vectors in two distinct cancer gene therapy approaches, TP53 gene replacement therapy and herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy. Moreover, we show that a deficiency of the primary receptor on the tumour cells limits the efficiency of adenovirus-mediated gene transfer in vivo. Since a number of studies have reported that primary cancer cells express only low levels of CAR, our results suggest that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realize the full potential of adenoviral vectors in the clinical setting.     

Gene transfer to carcinoma of the breast with fiber-modified adenoviral vectors in a tissue slice model system

Successful adenoviral (Ad) vector-mediated strategies for breast cancer gene therapy and virotherapy have heretofore been hindered by low transduction efficiency. This has recently been understood to result from a relative paucity of expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on primary tumor cells. To further investigate this issue, we evaluated the expression of CAR on breast cancer cell lines as well as primary breast cancer cells. With the exception of one patient sample, CAR expression was notably higher in the tumor cells from patients compared to CAR expression in the tumor cell lines. Furthermore, we explored CAR-independent targeting strategies to breast cancer tissue by exploring a panel of infectivity-enhanced Ad vectors, which contain CAR-independent targeting motifs for their utility in breast cancer gene therapy and virotherapy. These targeting motifs included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7), and were tested using the breast cancer tissue slice model, which is the most stringent substrate system available. Of all the tested tropism modified Ad vectors, Ad5/3 exhibited the highest transductional efficiency in breast cancer. These preclinical results suggest that Ad5/3 is the most useful modification to achieve higher clinical efficacy of breast cancer gene therapy and virotherapy.     

Coxsackievirus-adenovirus receptor expression in ovarian cancer cell lines is associated with increased adenovirus transduction efficiency and transgene expression

The expression of coxsackievirus-adenovirus receptor (CAR) and the integrins alpha(v)beta3 and alpha(v)beta5 was analyzed quantitatively (flow cytometry) and qualitatively (immunocytochemistry) in five human ovarian cancer cell lines (PEO1, PEO4, PEO14, SKOV-3, and OVCAR-3) and three control cell lines (293, HeLa, and CHO-K1). The transduction efficiencies were evaluated by adv/rsv-beta-Gal transduction followed by X-gal staining. The effects of 17beta-estradiol on cell growth, CAR and integrins alpha(v)beta3/5 expression, adenovirus transduction efficiency, and cell-killing efficacy of adv/rsv-tk plus ganciclovir were determined. The levels of CAR, integrin alpha(v)beta3, and integrin alpha(v)beta5 showed great variation between the cell lines. Whereas the expression of CAR appeared to be essential for and positively correlated with adenovirus transduction efficiency, the integrins alpha(v)beta3 and alpha(v)beta5 were not absolutely necessary for adenovirus transduction even though their presence may facilitate transduction. In PEO4 and PEO1 cells, proliferation was stimulated by 17beta-estradiol in a dose-dependent manner. In PEO4 cells, and much less pronounced in PEO1 cells, this was accompanied by an increase in CAR expression. The stimulation of CAR expression was paralleled by an increased transduction efficiency resulting in an increased cell-killing efficacy. Our data suggest that the expression of CAR is one of the most important prerequisites for successful adenovirus-mediated gene therapy of ovarian cancer.     

PCI-enhanced adenoviral transduction employs the known uptake mechanism of adenoviral particles

The development of methods for efficient and specific delivery of therapeutic genes into target tissues is an important issue for further development of in vivo gene therapy. In the present study, the physical targeting technique, photochemical internalization (PCI), has been used together with adenovirus. The combination of PCI and adenoviral transduction has previously been shown to be favorable compared to adenovirus used alone, and the aim of this study was to verify the role of the adenoviral receptors and identify the uptake pathway used by adenoviral particles in photochemically treated cells. All examined cell lines showed augmented transduction efficiency after PCI-treatment, with a maximum of 13-fold increase in transgene expression compared to conventionally infected cells. Blocking of CAR induced a complete inhibition of PCI-enhanced transgene expression. However, photochemical treatment managed to enhance the transduction efficiency of the retargeted virus AdRGD-GFP showing also that the virus-CAR interaction is not vital for obtaining a photochemical effect on adenoviral transduction. Blocking the alpha(V)-integrins reduced the gene expression significantly in photochemically treated cells. Subjecting HeLa cells expressing negative mutant-dynamin to light treatment after infection gave no significant increase in gene transfer, while the gene transfer were enhanced seven-fold in cells with wild-type dynamin. Furthermore, chlorpromazine inhibited photochemical transduction in a dose-dependent manner, whereas Filipin III had no effect on the gene transfer. In summary, the data presented imply that adenoviral receptor binding is important and clathrin-mediated endocytosis is the predominant uptake mechanism for adenoviral particles in photochemically treated cells.     

Cooperative effect of adenoviral p53 gene therapy and standard chemotherapy in ovarian cancer cells independent of the endogenous p53 status

Clinical adenoviral p53 gene therapy has been shown by us and others to inhibit tumor growth of ovarian cancer with endogenous mutant p53. This study was designed to test the cooperative antitumor effect of standard combination chemotherapy using paclitaxel and carboplatin together with adenoviral p53 gene transfer in the presence of wild-type and mutant p53. Seven ovarian cancer cell lines with mutant p53 and seven ovarian cancer cell lines with wild-type p53 were tested. An E1-deleted adenovirus type 5 expressing p53 (ACNp53) was used for p53 gene transfer. p53 gene transfer at 50% transduction efficiency significantly reduced IC50 of carboplatin chemotherapy up to 49-fold, of paclitaxel chemotherapy up to six-fold, and of paclitaxel/carboplatin chemotherapy up to 19-fold in the wild-type p53 cell line OV-MZ-5. Synergism between ACNp53 and chemotherapy calculated by median-effect analysis was found at low drug concentrations in all cell lines independent of the p53 mutational status. In conclusion, adenoviral p53 gene transfer significantly increased the sensitivity of ovarian tumor cells to paclitaxel, to carboplatin and/or to the combination of both.     

Antitumor effect of adenovirus-mediated Bax gene transfer on p53-sensitive and p53-resistant cancer lines

Antitumor effects of the proapoptotic Bax gene have been evaluated in vitro and in vivo by a binary adenovirus system expressing the human Bax gene. Overexpression of the Bax gene in cultured cell lines from human lung carcinoma results in caspase activation, apoptosis induction, and cell growth suppression. Intratumoral injection of adenovirus vector expressing the Bar gene suppressed growth of human lung cancer xenografts established in nude mice. Histological examination of tumors from mice treated with the Bax gene demonstrated high levels of Bax expression and extensive apoptosis in tumors. In comparison with the treatment by an adenoviral vector expressing human p53, the Bax gene can effectively suppress tumor growth in both p53-sensitive and p53-resistant human lung carcinoma cell lines. Toxicity was not detected in liver and other systems in animals treated intralesionally with the Bax gene. Therefore, our results suggest that the Bar gene may be useful in cancer treatment.     

Light-induced adenovirus gene transfer, an efficient and specific gene delivery technology for cancer gene therapy

A main issue for further clinical progress of cancer gene therapy is to develop technologies for efficient and specific delivery of therapeutic genes to tumor cells. In this work, we describe a photochemical treatment that substantially improves gene delivery by adenovirus, one of the most efficient gene delivery vectors known. Transduction of two different cell lines was studied by microscopy, flow cytometry, and an enzymatic assay, employing a beta-galactosidase-encoding adenovirus. The photochemical treatment induced a >20-fold increase in gene transduction, compared with conventional adenovirus infection, both when measured as the percentage of cells transduced, and when measured as the total beta-galactosidase activity in the cell population. The effect was most pronounced at lower virus doses, where in some cases the same transduction efficiency could be achieved with a 20 times lower virus dose than with conventional infection. Photochemical treatments are already in clinical use for cancer therapy, and generally are very specific and have few side effects. The photochemical internalization technology described thus has a clear potential for improving both the efficiency and the specificity of gene delivery in cancer gene therapy, making it possible to achieve efficient site-specific in vivo gene delivery by adenoviral vectors.     

Coxsackievirus adenovirus receptor expression predicts the efficiency of adenoviral gene transfer into non-small cell lung cancer xenografts.

PURPOSE: Current paradigms postulate that inefficient adenoviral (Ad) gene transfer is a consequence of poor Coxsackievirus adenovirus receptor (CAR) expression in tumors in vivo. To test whether exuberant CAR expression alone is sufficient to mediate efficient Ad gene transfer, we compared Ad gene transfer efficiency in a panel of non-small cell lung cancer (NSCLC) cell model systems in which we systematically measured CAR expression in vitro and in vivo. EXPERIMENTAL DESIGN: NSCLC cells were selected for study on the basis of (a) differences in Ad transduction, (b) identical requirements for growth in vitro, (c) capacity to grow as xenografts in immunocompromised mice, and (d) similar amounts of alpha(v) integrin expression as measured by flow cytometry. CAR expression and Ad transduction profiles of these NSCLC cells were generated in vitro and in vivo. RESULTS: Ad transduction efficiency of NSCLC cells in vitro can be directly related to CAR expression at both the mRNA and protein level. CAR expression in vitro favorably predicts a comparable pattern of expression in transplanted NSCLC xenografts in vivo. Xenografts generated from NSCLC cells exhibiting increased CAR expression showed evidence of higher Ad gene transfer, although the efficiency of transduction was reduced compared with in vitro measurements. Thus, in NSCLC cells with high basal expression of CAR, Ad vector doses that enabled uniform transduction in vitro achieve a gene transfer efficiency ranging from 10% to 70% after a single intratumoral injection in the xenografts. CONCLUSIONS: These studies indicate CAR expression is predictive for more efficient gene transfer into NSCLC cells in vitro and in vivo but is not sufficient to achieve uniform transduction by Group C Ad vectors in vivo.