Growth Regulated Oncogene-α expression by murine squamous cell carcinoma promotes tumor growth, metastasis, leukocyte infiltration and angiogenesis by a host CXC Receptor-2 dependent mechanism

Abstract

Growth Regulated Oncogene-α (GRO-α) is an autocrine growth factor in melanoma and is a member of the C-X-C family of chemokines which promote chemotaxis of granulocytes and endothelia through binding to CXC Receptor 2. We found previously that variants of murine squamous cell carcinoma PAM 212 which grow and metastasize more rapidly in vivo constitutively express increased levels of murine GRO-α, designated mGRO-α, or KC. We have examined the possible role of mGRO-α expression in malignant progression of squamous cell carcinoma PAM 212 in homologous BALB/c and BALB CXC Receptor-2 deficient mice. Transfection of the PAM 212 cell line which exhibits low expression of GRO-α and malignant potential with a pActin-KC vector encoding mGRO-α enabled isolation of PAM-KC expressing cell lines. These PAM-KC transfectants displayed an increased rate of growth and metastasis in BALB/c mice, similar to the highly malignant phenotype observed in spontaneously occurring metastatic variants. Furthermore, the PAM-KC tumors showed an increase in infiltration of host leukocytes and CD31+ blood vessels, consistent with increased CXC chemokine activity. The increased growth of PAM-KC cells was attenuated in CXCR-2 deficient mice, indicating that the increased growth was dependent in part upon host cells responsive to the CXC chemokine. Together, these results show that a CXC chemokine such as GRO-α can promote malignant growth of murine squamous cell carcinoma by a host CXCR-2 dependent pathway.

Introduction

Squamous cell carcinomas (SCC) may arise in the skin, upper aerodigestive tract, lung and cervix, and are a significant cause of cancer morbidity and mortality in the US and worldwide. Development of SCC and other cancers is often associated with increased leukocyte infiltration and angiogenesis (Pak et al., 1995; Gasparini et al., 1993), and increased density of leukocytes and blood vessels has been associated with a higher risk of progressive tumor growth, metastasis, and death in patients (Gasparini et al., 1993; Young et al., 1997) and in experimental animal models (Gutman et al., 1994; Seung et al., 1995). Identification of the factor(s) that induce these inflammatory and angiogenesis responses, and their role in promotion of malignant progression, has been the subject of considerable interest to investigators studying SCC and other cancers (Schreiber and Rowley, 1999).

We found previously that variants of murine squamous cell carcinoma PAM 212 which grow and metastasize more rapidly in vivo differentially express increased levels of murine GRO-α (mGRO-α), originally named KC (Dong et al., 1997). GRO-α is an autocrine growth factor identified in melanoma (Balentien et al., 1991), and is a member of the C-X-C family of chemokines which promote chemotaxis of granulocytes and endothelia (Strieter et al., 1996). We and others have found that human SCC also produce C-X-C chemokines with potential proinflammatory and proangiogenic activity, including IL-8 (Chen et al., 1998, 1999; Cohen et al., 1995; Smith et al., 1994) and GRO-α (Loukinova, unpublished results). mGRO-α shares major sequence homology and leukocyte chemotactic activity (Oquendo et al., 1989; Bozic et al., 1995) with both human GRO-α and IL-8, for which independent murine counterparts have not been identified. Human IL-8 and GRO-α have been reported to induce chemotaxis of neutrophils and neovascular endothelial cells (Smith et al., 1994; Strieter et al., 1996). While mGRO-α has been shown to induce neutrophil chemotaxis (Bozic et al., 1995), the angiogenic activity of murine GRO-α has not been reported. The effects of these C-X-C chemokines on leukocyte chemotaxis and angiogenesis are consistent with the distribution of C-X-C receptor-2 (CXCR2), to which they have been reported to bind. CXCR2 is expressed on neutrophils and endothelia (Strieter et al., 1996).

We have recently shown that expression of C-X-C chemokines GRO-α and IL-8 in murine and human SCC is induced by constitutive activation of transcription factor Nuclear Factor-κB/Rel A (Dong et al., 1999; Duffey et al., 1999; Ondrey et al., 1999), and that inhibition of expression of cytokine expression by inhibition of NF-κB can inhibit growth of human SCC xenografts in SCID mice (Duffey et al., 1999). Others have shown that the growth of non-small cell lung and prostate cancer xenografts in immunodeficient SCID mice can be inhibited using antisera against IL-8 (Smith et al., 1994), or IL-8 and GRO-α (Moore et al., 1999). The decrease in growth correlated with decreased vessel density, suggesting that host angiogenesis responses may be important in tumorigenesis. However, the lack of complete homology between human and murine cytokines and their receptors, as well as adhesion molecules, may confound the interpretation of studies of cytokine-mediated growth regulation in xenograft models described above by us and others. Moreover, the limited growth and development of metastasis of human tumors in xenograft models, which presumably arise in part due to these incompatibilities, may limit our understanding of the extent to which these chemokines contribute to advanced tumor progression and metastasis.

We previously developed a syngeneic murine model of SCC in our laboratory that has provided an opportunity to examine the expression of cytokines and chemokines during the early and late extremes of tumor progression in a homologous system (Chen et al., 1997; Smith et al., 1998; Dong et al., 1997, 1999). The model consists of the spontaneously transformed keratinocyte line PAM 212, and lines isolated from rare PAM metastases which spontaneously arose following inoculation of PAM 212 in mice. We found that transformed PAM 212 keratinocytes exhibit low tumorigenicity and metastatic potential in association with low production of cytokines, while PAM metastases rapidly grow and metastasize, and express a repertoire of cytokines similar to that detected in human tumors (Smith et al., 1998). The cytokines expressed by these metastatic variants included IL-1α, IL-6, GM-CSF and mGRO-α (KC). We found that KC mRNA and protein was uniformly expressed at high concentration by 15/15 cell lines reisolated from lymph node and lung metastases of the murine SCC line PAM 212 (Smith et al., 1998). These reisolated metastatic cells, exhibited an increased potential for growth and metastasis in vivo when compared with the low KC expressing PAM 212 cell line (Chen et al., 1997; Dong et al., 1997). Conversely, clones of PAM 212 which expressed no detectable KC by ELISA grew even more slowly than the low KC expressing PAM 212 cell line (Smith et al., 1998).

The presence of a structural relationship between murine and human GRO-α and IL-8 and the correlation of expression of these chemokines with tumor progression led us to examine the possible cause and effect relationship between mGRO-α production by tumor cells and increased malignant behavior in our homologous murine model. We show here that an increase in constitutive expression of mGRO-α in the low mGRO-α-expressing parental cell line PAM 212 results in increased growth and metastasis of SCC in homologous BALB/c mice. Expression of GRO-α is associated with increased density of leukocytes and blood vessels. Tumor growth was attenuated in BALB/c mice deficient in expression of CXCR-2, indicating that both CXC chemokine and CXC specific host responses contribute to increased malignant behavior.

Results

Increased expression of KC mRNA and protein by SCC reisolated from lymph node and lung metastases

We previously detected increased expression of KC mRNA encoding murine GRO-α by differential display in SCC reisolated from lymph node and lung following metastatic tumor progression of the in vitro transformed keratinocyte line PAM 212 (Dong et al., 1997). Figure 1a shows a Northern blot analysis confirming that KC mRNA expression is increased in representative PAM LY and LU cell lines reisolated from lymph node and lung metastases when compared with normal BALB/c and transformed PAM 212 keratinocytes. ELISA analysis for KC protein in supernatants from the same cultures in Figure 1b shows that PAM LY and LU tumors secrete 3000 pg protein/106 cells. The increase in KC protein secretion by SCC metastases PAM LY 1, -2, and PAM LU1 is consistent with the differences in mRNA expression observed. A similar increase in secretion of KC was observed in 9/9 cell lines from lymph node and 6/6 cell lines from lung metastases (Van Waes et al., 1999). We previously confirmed that the KC mRNA and protein detected in the metastatic reisolates was expressed by malignant keratinocytes positive for keratin K6 and 14 (Chen et al., 1997).

Figure 1
figure1

Expression of KC mRNA (a) and protein (b) by primary BALB/c keratinocytes, parental PAM 212 and metastatic cell lines PAM LY1, 2 and LU1. Cell line PAM 212 is an SCC line derived following spontaneous transformation of BALB/c keratinocytes in vitro, and PAM LY and LU cell lines were derived from lymph node and lung metastasis of PAM 212 in BALB/c mice, as described in Materials and methods. (a) Northern blot analysis is shown comparing KC mRNA expression between cell lines (upper panel), using constitutive expression of actin as a control (lower panel). (b) ELISA showing concentration of immunoreactive KC protein secreted after 24 h per 106 cells, as mean±s.e.m. Expression of KC mRNA (c) and protein (d) by parental PAM 212 and transfectant cell lines PAM-Vector and PAM-KC. (c) Northern blot analysis is shown comparing KC mRNA expression between cell lines (upper panel), using constitutive expression of actin as a control (lower panel). (d) ELISA showing concentration of immunoreactive KC protein secreted after 24 h per 106 cells, as mean±s.e.m.

Expression of KC in the PAM 212 cell line following gene transfer

KC cDNA was cloned, sequenced and placed under control of the actin promoter in a vector (pActin-KC) for expression studies, as described in Materials and methods. Vector containing KC or control vector lacking the insert was transfected into the low KC expressing parental PAM 212 cell line. Transduced cells were then selected in G418, cloned by limiting dilution, and selected for expression of KC by specific ELISA analysis of supernatants. Preliminary experiments showed that PAM-KC clones expressing KC at concentrations between 100–8000 pg/ml and PAM-Vector clones expressing KC at levels 100 pg/ml above the levels expressed by parental PAM 212 cell line population showed a comparable increase in the rate of proliferation of approximately 20% in MTT assay, providing evidence that increased concentrations of KC can stimulate proliferation of SCC through an autocrine mechanism that is similar to that observed for melanoma (Balentien et al., 1991). To examine for any additional effects differences in KC expression had upon growth due to host dependent mechanisms, four PAM-KC and four PAM-Vector clones which demonstrated comparable intrinsic proliferative rates in MTT assay were pooled to control for differences in growth due to differences in autocrine stimulation and clonal heterogeneity. Figure 1c,d shows a comparison of KC mRNA and protein secretion in parental PAM 212, pooled PAM-Vector and PAM-KC cell lines. Figure 1d confirms that PAM-KC cells secrete 3000 pg protein/106 cells, while PAM 212 and PAM-Vector are low KC producers. We confirmed that selection and expression of KC did not result in detectable expression of cytokines IL-1α, IL-6 or GM-CSF (data not shown) which can also be detected in spontaneously occurring PAM metastatic variants (Smith et al., 1998).

Growth of PAM 212, PAM-Vector and PAM-KC transfected cells in vitro and in vivo

To compare the intrinsic proliferative rates of the cell lines, growth of PAM 212, PAM-Vector and PAM-KC transfected cell lines in vitro was compared by MTT assay. Figure 2a shows that there is no significant difference in growth between PAM 212, PAM-Vector and PAM-KC transfected cells in vitro. The similarity in endogenous proliferative rate among the pooled lines was consistent with our comparison of individual PAM-KC and PAM-Vector clones in preliminary experiments, and results showing that parental PAM 212 and metastatic PAM LY cells which secrete KC grow at similar rates in vitro (Chen et al., 1997).

Figure 2
figure2

Growth of parental PAM 212, PAM-Vector and PAM-KC cells (a) in vitro and (b) in vivo. (a) MTT assay was performed comparing growth over 5 days of PAM 212, PAM-Vector and PAM-KC cells. 5×103 cells were cultured in 96 well plates and the density of quadruplicate samples was determined following addition of MTT as described in Materials and methods. (b) Growth of PAM 212, PAM-Vector and PAM-KC cells in syngeneic BALB/c mice was determined by comparing L×W of tumors which developed following inoculation of 5×106 cells s.c. of each cell line in the flank, using 10 mice per group. MTT OD and tumor area are reported as means±s.e.m.

To determine if increased expression of KC may promote growth of SCC by a host dependent mechanism, growth of PAM 212, PAM-Vector and PAM-KC transfected cells was compared in syngeneic BALB/c mice in vivo. Figure 2b shows that in vivo, PAM-KC cells grow more rapidly than the parental PAM 212 or PAM-Vector transfected cell line, even while PAM-KC cells grew at a similar rate in vitro. To confirm these results, we conducted another transfection and selected independent PAM-KC and PAM-Vector lines for comparison, as before. In the independent experiment shown in Figure 3a, the PAM-KC expressing cell line grew more rapidly relative to the PAM-Vector cell line in BALB/c mice, reducing the likelihood that the difference observed in growth of PAM-KC tumors arose due to chance differences in heterogeneity among selected clones. Furthermore, increased growth was observed in both immune competent (Figure 3a) and T lymphocyte-deficient BALB athymic nude mice (Figure 3b), indicating that the difference in growth is independent of differences in T lymphocyte dependent immunity or tolerance. These results are consistent with our previous results showing that increased growth of spontaneously arising PAM LY metastases that secrete KC in vivo depends on a T lymphocyte-independent host mechanism (Chen et al., 1997).

Figure 3
figure3

Comparison of growth of PAM-Vector and PAM-KC cells in (a) BALB/c and (b) T cell deficient congenic BALB nu/nu athymic mice. PAM-Vector and PAM-KC cells were obtained following an independent transfection and selection in G418. Growth of PAM-Vector and PAM-KC cells in syngeneic BALB/c and congenic BALB nu/nu athymic mice was determined by comparing L×W of tumors which developed following inoculation of 5×106 cells s.c. of each cell line in the flank, using 10 mice per group. Tumor areas are reported as means±s.e.m.

Decreased growth of PAM-KC tumors in BALB CXCR2 deficient transgenic mice

mGRO-α (KC) was previously shown to be a ligand for the IL-8B receptor, now designated CXCR2 (Strieter et al., 1996). To explore whether growth of PAM-KC is promoted by KC-induced host responses involving CXCR2, we inoculated BALB/c and BALB mice deficient for CXCR2 expression with PAM-KC. Figure 4a shows that growth of PAM-KC is markedly inhibited in CXCR2 deficient mice relative to normal BALB/c controls. The reduced rate of growth of PAM-KC in CXCR2 deficient mice in Figure 4a was similar to the growth of the low KC producing parental PAM 212 cell line in both BALB/c and CXCR2 deficient mice (Figure 4b). These data provide evidence that the increase in growth of PAM-KC observed in the experiments above are due specifically to a CXC chemokine-mediated host mechanism, and not other tumor or host related differences resulting from heterogeneity in the PAM-KC and PAM-Vector cell lines. We conclude that chemokine KC (mGRO-α) contributes to malignant growth of PAM-KC through a CXCR2 host-dependent mechanism.

Figure 4
figure4

Comparison of growth of PAM-KC and parental PAM 212 cells in BALB/c and congenic BALB CXCR2 deficient mice. (a) Growth of PAM-KC cells was reduced in CXCR2 deficient mice relative to BALB/c mice, (P<0.05). (b) Growth of low GRO-α/KC producing PAM 212 cells was similar in both CXCR2 deficient mice and BALB/c mice (P>0.05). Area was determined by comparing L×W of tumors which developed following inoculations of 5×106 PAM-KC cells s.c. in the flank, using five mice per group. Tumor areas are reported as means±s.e.m.

Increased incidence of lung metastases in mice bearing PAM-KC tumors

To determine if expression of KC and increased growth is linked with an increased incidence of SCC lung metastases in mice, the number of mice with lung metastases was enumerated in BALB/c recipients bearing PAM 212, PAM-Vector control and PAM-KC tumors 6 weeks following inoculation with 5×106 tumor cells. Figure 5 summarizes the results of four independent experiments comparing the incidence of lung metastases. An increase in the number of animals with lung metastases was observed in recipients of PAM-KC cells compared with PAM 212 or PAM-Vector transfected cells (P<0.05). The proportion of PAM-KC bearing mice with lung metastases approached the proportion observed in mice inoculated with the highly metastatic KC producing PAM LY2 line (Figure 5). No significant difference in the highly variable range in metastases burden (1 to >100 nodules/animal) was observed between BALB/c mice bearing PAM-KC and PAM LY2. In the study of PAM-KC in CXCR2 deficient mice, no pulmonary metastases were detected.

Figure 5
figure5

Incidence of pulmonary metastases in BALB/c mice bearing PAM 212, PAM LY2, PAM-Vector and PAM-KC tumors. Percentage of BALB/c mice with visible metastases following staining with Bouin's solution was determined 6 weeks following inoculation of 5×106 cells s.c. of each cell line in the flank, using 10 mice per group. Incidence of metastases are reported as mean±s.e.m. of four independent experiments. Differences between PAM-Vector and PAM KC were significant, (P<0.05)

Increase in infiltration of leukocytes and vessel density in KC expressing PAM metastases and PAM-KC tumors

Inflammatory and angiogenesis responses are commonly observed in SCC (Pak et al., 1995; Gasparini et al., 1993; Young et al., 1997), and human GRO-α has been shown to promote an increase in neutrophil chemotaxis and vessel density (Strieter et al., 1996). We examined PAM 212 tumors and PAM metastases for evidence of inflammatory and vascular changes, since we showed in Figure 1b that PAM 212 expresses low levels of KC while PAM metastases express KC at increased levels. Figure 6a shows a representative region of a PAM 212 tumor, which demonstrates relatively little inflammation or vascularity. Figure 6b shows a PAM lung metastasis. The PAM metastasis is associated with increased accumulation of intravascular and perivascular leukocytes (Figure 6b, arrows), consistent with expression of a leukocyte chemotactic factor. To determine if increased cellular leukocyte inflammatory response observed in SCC may be specifically associated with expression of KC, we compared the histology of PAM-Vector and PAM-KC tumors (Figure 6c,d). While PAM-Vector tumors again exhibit relatively little inflammation or vascularity (Figure 6c,e), an increase in intravascular accumulation and perivascular infiltrating cells is observed in PAM-KC tumors (Figure 6d,f, arrows). Polymorphonuclear leukocytes and mononuclear cells are visible. An apparent increase in vascularity between PAM-Vector and PAM-KC tumors is also suggested by the presence of more numerous, enlarged, vascular spaces (compare Figure 6e,f, arrows). To determine if expression of murine KC is associated with an increase in vascular density in SCC, we compared the density of PECAM (CD31) positive vessels in tumor from recipients of PAM-Vector and PAM-KC transfected cells. Figure 6g,h shows that the density of vessels is increased in a PAM-KC tumor relative to a PAM-Vector control tumor. The increase in leukocyte infiltration and vascular density in PAM-KC tumors in BALB/c mice was not observed in CXCR2-deficient mice (not shown). To compare the density of leukocytes and vessels, we counted the number of perivascular infiltrating polymorphonuclear leukocytes, mononuclear cells and CD31+ vessels in PAM-Vector and PAM-KC tumors in BALB/c mice. Figure 7a shows that PAM-KC tumors exhibit an increase in density of both polymorphonuclear leukocytes and monocytes in PAM-KC tumors. Figure 7b shows that PAM-KC tumors also exhibit and increase in vessel density. These results are consistent with the hypothesis that KC can promote increased inflammation and angiogenesis observed in SCC.

Figure 6
figure6

Histology of parental PAM 212, metastatic PAM, PAM Vector and PAM-KC tumors. Hematoxylin and eosin stained specimens: (a) Representative region of a PAM 212 tumor, demonstrating relatively little inflammation or vascularity. (b) PAM metastasis in the lung associated with increased accumulation of intravascular and perivascular leukocytes (arrows). (c) PAM-Vector and (d) PAM-KC tumors, demonstrate an increase in accumulation of intravascular and perivascular infiltrating cells with expression of KC (arrows). Polymorphonuclear leukocytes and mononuclear cells are visible. (e) PAM-Vector and (f) PAM-KC tumors. An apparent increase in vascularity between PAM-Vector and PAM-KC tumors is also suggested by the presence of more numerous, enlarged, vascular spaces (compare Figure 6e,f, arrows). CD31 (PECAM) immunoperoxidase staining of (g) PAM-Vector and (h) PAM-KC tumors. (g), (h) show an increase in the density of vessels in a PAM-KC tumor relative to a PAM-Vector control tumor

Figure 7
figure7

Density of infiltrating leukocytes and CD31+ vessels in PAM-Vector and PAM-KC tumors. (a) The density of infiltrating polymorphonuclear and mononuclear leukocytes in PAM-Vector and PAM-KC tumors was determined from the mean number of cells in three high power fields surrounding representative vessels,±s.e.m. A significant increase in PMNs (P=0.016) and monocytes (P=0.03) in PAM-KC tumors was detected. (b) The density of vessels in PAM-Vector and PAM-KC tumors was determined by counting the number of vessels per high power field in the area of highest density at the tumor periphery, and represented as the mean±s.e.m. A significant increase in CD31+ vessels (P=0.03) in PAM-KC tumors was detected

Discussion

We have shown that expression of CXC chemokine mGRO-α (KC) by squamous carcinoma cells increases tumor growth and metastasis in a model in which cytokines and receptors expressed by tumor cells and the host are homologous. Expression of mGRO-α at high levels in the low mGRO-α expressing SCC line PAM 212 following gene transfer increased growth and metastases of PAM-KC tumors in independent experiments (Figures 2,3,4,5). Thus, the malignant phenotype resulting from expression of mGRO-α in the PAM-KC transfectants was similar to that observed in the highly malignant variants of PAM that had been derived from experimental metastases and also constitutively expressed elevated levels of mGRO-α mRNA and protein (Chen et al., 1997; Van Waes et al., 1999). When PAM-Vector and PAM-KC lines with similar proliferative rates were used to control for the effects of autocrine stimulation, the increase in mGRO-α expression was found to promote an increase in growth of SCC through a host dependent response, as well as via autocrine stimulation of proliferation, which has been seen in melanoma. We confirmed this hypothesis by showing that host cells which express the CXCR2 receptor are required to enhance the malignancy of mGRO-α secreting tumor cells, because growth of PAM-KC tumor cells was attenuated in BALB CXCR2 deficient mice (Figure 4). These results indicate that the increase in tumor growth was due specifically to a CXC chemokine mediated host mechanism, and not likely to be due to other cellular or host dependent differences resulting from nonspecific effects of transfection, selection, or production of other non-CXC cytokines. The increase in mGRO-α expression was associated with an increase in vascular density and infiltration of polymorphonuclear and mononuclear leukocytes in PAM-KC tumors. Increased vascular density and infiltration of polymorphonuclear and mononuclear leukocytes has previously been associated with increased growth, metastasis and a decrease in prognosis in human and murine tumors (Pak et al., 1995; Gasparini et al., 1993; Young et al., 1997; Gutman et al., 1994; Seung et al., 1995). These results provide evidence that the increased expression of CXC chemokines such as mGRO-α by SCC can induce host responses which provide a selective advantage for malignant growth in vivo.

The increase in density of microvasculature and leukocytes observed in PAM-KC tumors is consistent with that of host responses induced by those CXC chemokines which share the amino acid motif E-L-R-C-X-C, such as mGRO-α, human GRO-α and IL-8 (Strieter et al., 1996). Human IL-8 and GRO-α have been reported to induce angiogenesis and neutrophil chemotaxis in animals in vivo (Strieter et al., 1996). Further, the growth of human non-small cell lung cancer cells expressing IL-8 may be inhibited in SCID mice by anti-IL-8 antisera (Smith et al., 1994). Inhibition of growth of human prostate cancer cell lines expressing both GRO-α and IL-8 required inhibition by antisera against both of these CXC chemokines (Moore et al., 1999). We have shown that inhibition of expression of CXC chemokine IL-8 and other cytokines by inhibition of transcription by Nuclear Factor-κB inhibits growth of human SCC xenografts in SCID mice (Duffey et al., 1999). However, the presence of multiple differences in homology that result in incompatibilities between cytokine-receptor networks, and lack of metastatic tumor progression, limit the conclusions which may be drawn from studies of human–animal xenografts. The present study conducted in a syngeneic murine SCC model provides direct evidence that expression of a CXC chemokine, such as GRO-α, can also promote metastatic tumor progression.

While murine SCC express GRO-α and do not express IL-8, we have detected expression of both IL-8 and GRO-α in human SCC (Chen et al., 1998, 1999; Loukinova, unpublished observations). Thus, as has been observed with human prostate cancer, human SCC may express more than one E-L-R-C-X-C chemokine with potential proinflammatory and proangiogenic activity. It therefore seems possible that the activity of more than one CXC chemokine may need to be blocked by anti-cytokine therapy in order to arrest growth and metastasis of human SCC. Several chemokines containing an E-L-R-C-X-C amino acid motif have been reported to bind CXCR2 (IL-8RB) (Strieter et al., 1996), and inhibition of CXCR2 may therefore be an important target for anti-chemokine therapy of SCC and other cancers which produce one or more of these chemokines.

It seems likely that expression of CXC chemokines and other angiogenesis factors also contribute to the growth of PAM tumors through mechanisms involving autocrine stimulation, and angiogenesis. The slow outgrowth of PAM-KC and PAM 212 in CXCR2 deficient mice suggests that PAM tumors may produce enough KC and other factors to induce growth (Figure 4a,b). We found that KC does induce a 20% increase in proliferation, providing evidence for autocrine as well as host CXCR2 dependent stimulation of growth (data not shown). A 20% increase in growth was observed over a range of 100–8000 pg/ml, indicating that the rate of proliferation of PAM 212 due to autocrine stimulation by KC is already near maximum. This evidence for autocrine stimulation is consistent with that observed previously in melanoma. We have reported that human SCC produce a repertoire of other cytokines with putative proangiogenic activity, including vascular endothelial growth factor and basic fibroblast growth factor (Chen et al., 1998), which could also contribute to the growth of PAM 212 and PAM-Vector cells. Alternatively, decreased expression of angiostatic factors may promote host response to angiogenic factors. Strieter and colleagues (1996) have hypothesized that increased neoangiogenesis may result not only from an increase in ELR-positive CXC chemokine expression, but from a decrease in ELR-negative CXC chemokine expression, since ELR negative chemokines such as Interferon Inducible Protein-10, have been reported to be angiostatic. It will therefore be important to determine whether increased expression of other angiogenesis factors or decreased expression of angiostatic cytokines may also be important in pathogenesis and therapy of SCC.

We show here that mGRO-α is important in inducing angiogenesis and inflammatory responses and aggressive behavior of SCC. While the potential advantage of increased vascular supply has been well accepted (Weidner and Folkman, 1996), the mechanisms by which inflammatory responses may promote malignant behavior have not been fully defined. Seung, Pekarek and colleagues have shown that granulocytes can promote growth of UV induced skin tumors (Seung et al., 1995; Pekarek et al., 1995), and Young and colleagues have shown that CD34+ cells of the granulocyte lineage induced by SCC can promote both growth, metastasis and immune suppression (Pak et al., 1995; Young et al., 1997). GRO-α and IL-8 are chemoattractants which are effective in recruiting granulocytes and macrophages. Macrophages and other leukocytes may promote tumor angiogenesis through the release or activation of angiogenesis substances (Gutman et al., 1994; Schreiber and Rowley, 1999). Since the chemokines and cytokines regulating the chemotaxis and activation of these stromal leukocytes in cancer have not previously been defined, the identification of GRO-α as an important promoter of metastatic tumor progression may help in defining the function of these leukocytes, and new targets for therapy.

Materials and methods

SCC cell lines and culture conditions

Non-transformed keratinocytes were cultured from the skin of male BALB/c neonates, as described (Yuspa et al., 1980). The PAM 212 cell line was established from neonatal BALB/c keratinocytes, which were spontaneously transformed and passaged in vitro (Yuspa et al., 1980). PAM LY and LU cell lines were isolates from rare lymph node (LY-1, LY-2) and lung (LU-1) metastases that formed following subcutaneous inoculation of 5×106 PAM 212 cells in BALB/c mice, and have previously been shown to grow more aggressively and form greater lymph node and lung metastases than the parental PAM 212 cell line (Chen et al., 1997). PAM LY and LU tumors have previously been shown to express keratin and integrin markers consistent with those expressed by primary and transformed PAM 212 keratinocytes (Chen et al., 1997). All SCC cell lines were grown in EMEM with 10% fetal calf serum, penicillin, and streptomycin in a humidified atmosphere of 5% CO2 at 37°C. All cells were demonstrated to be free of Mycoplasma contamination by ELISA assay.

Construction of KC expression plasmid

To clone the full-length coding sequence for KC, 5′ RACE was performed using a Marathon cDNA Amplification kit from ClonTech (PT1115-1, ClonTech, Palo Alto, CA, USA) following the manufacturer's recommendations. Briefly, 1 μg of poly(A)+ RNA isolated from PAM LY-1 was used to make double-stranded cDNA. After ligation of cDNA with adaptors provided in the kit, PCR was performed with primer AP-1 in the kit and KC-specific primer KC3-1. The sequence of KC3-1 is 5′-TATAGTGGTGTCAGAAGCCAGCGTTCACC-3′. PCR conditions were as follows: 1 min at 94°C (one cycle), 30 s at 94°C, 4 min at 72°C (five cycles), 30 s at 94°C, 4 min at 70°C (five cycles), and 30 s at 94°C, 4 min at 68°C (20 cycles). The specific cDNA product was then amplified by a nested PCR under the same conditions with primer AP-2 in the kit and KC3-2 (5′-AGAGCAGTCTGTCTTCTTTCTCCGTTAC-3′). A single 400-bp product was obtained, gel purified, and cloned into pCR2.1 vector (Invitrogen) to yield pCR-KC. DNA sequencing of pCR-KC from both ends confirmed that RT–PCR product contained the full-length coding sequence of KC without mutations. The cDNA insert containing KC coding sequence was isolated from pCR-KC by digesting with SmaI and EcoRI, and transferred into pHbAPro-1neo by blunt end ligation to generate pActin-KC.

Transfection of PAM 212 cells

PAM 212 cells were grown in EMEM with 10% fetal calf serum and penicillin/streptomycin. pActin-KC and pActin only were transfected with Lipofectamine according to the manufacturer's recommended protocol (Life Technology, Inc.). The cells were cultured in EMEM with 10% fetal calf serum and penicillin/streptomycin 48 h and selected in G418 for 2 weeks. The G418 selected colonies were tested for KC production by ELISA.

RNA preparation and Northern blot analysis

Total RNA from cell lines was isolated using Trizol reagent (Life Technologies, Inc.). Twenty μg of RNA from each sample was denatured in formaldehyde and formamide at 65°C for 15 min, and subjected to electrophoresis in a 1.2% agarose formaldehyde denaturing gel. RNA was transferred to a nylon membrane (Hybond, Amersham LIFE SCIENCE) by capillary transfer with 20×SSC. After UV crosslinking, the membrane was prehybridized for 1 h at 68°C in QuikHyb solution (Stratagene). GRO-α/KC cDNA was labeled with [32P]dCTP using a random-prime DNA labeling kit Prime-It RmT (Stratagene). The filters were hybridized to probe overnight at 68°C using standard conditions, followed by autoradiography overnight at −80°C.

ELISA determination of KC production by PAM cell lines

Commercially available ELISA kits were used to quantitate the level of KC produced by PAM cell lines and PAM transfectants (Endogen, Woburn, MA, USA). Culture medium was replaced when cell lines grown in T25 reached 60–80% confluency. Supernatants were collected and cell counts were performed after 24 h. The assay was performed according to the instructions of the manufacturer. The optical density of the calorimetric reaction was read by multichannel spectrophotometer at a wavelength of 450 nm (EL 311, Biotec, and Winooski, VT, USA). The quantity of cytokine in culture was determined by comparison with recombinant KC standards in pg/106 cells/24 h.

Cell growth assay

Cells were seeded at a concentration of 5×103/well in 200 μl culture medium into 96 well tissue culture plates. After incubation of cultures for 1, 3, and 5 days at 37°C and 5% CO2, 100 μl of medium from each well was removed and 10 μl of MTT labeling solution was added to each well. After 4 h incubation at 37°C and 5% CO2 100 μl of solubilization solution was added. After 18 h incubation, the density of cells was quantified using a scanning multiwell spectrophotometer at a wavelength of 570 nm.

Tumor growth and metastasis studies

Six- to eight-week-old male BALB/c and BALB nu/nu mice were obtained from the National Cancer Institute, Frederick, Maryland, USA and BALB CXCR2 deficient transgenic mice were obtained from Jackson Laboratories, Bar Harbor, ME, USA. Mice were maintained with sterile food, bedding and caging in the NIH animal facility, and studies were performed under an NIH Animal Care and Use Committee approved protocol 802-97. PAM 212, PAM-Vector and PAM-KC transfected cell lines were harvested with trypsin/EDTA, washed three times with EMEM and resuspended at a concentration of 5×106 cells/200 μl EMEM. Mice were inoculated subcutaneously with 200 μl of cell suspension in the right flank. Tumor volumes were determined twice a week. Tumor area was calculated using the formula: tumor area=tumor width×tumor length. After tumor area reached approximately 2 cm2 the mice were euthanized by CO2 inhalation. The lungs were removed and stained in Bouin's solution for 5 days, and lungs with visible metastatic colonies were counted.

Determination of tumor infiltrating leukocyte and vessel density

Tumors were isolated and frozen in OCT compound. 5–8 μm frozen sections were obtained. Sections were fixed in formalin and stained by hematoxylin and eosin, or fixed in methanol:acetone and stained with antibody specific for CD31 (PECAM, Pharmingen, Inc.), followed by immunoperoxidase (Vector Labs). The density of infiltrating polymorphonuclear and mononuclear leukocytes in PAM-KC and PAM-Vector tumors was determined by counting the number of cells in three high power fields surrounding representative vessels, and determining the mean±s.d. The density of vessels in PAM-KC and PAM-Vector tumors was determined by counting the number of vessels per high power field in the area of highest density in the tumor periphery.

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Acknowledgements

We thank Drs James Battey and Stuart Yuspa for helpful discussions during the course of these studies and comments on the manuscript. The study was supported by NIDCD intramural research project Z01-DC-00016 (C Van Waes) and NCI grant CA-22677 (H Schreiber).

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Loukinova, E., Dong, G., Enamorado-Ayalya, I. et al. Growth Regulated Oncogene-α expression by murine squamous cell carcinoma promotes tumor growth, metastasis, leukocyte infiltration and angiogenesis by a host CXC Receptor-2 dependent mechanism. Oncogene 19, 3477–3486 (2000). https://doi.org/10.1038/sj.onc.1203687

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Keywords

  • GRO-α
  • KC
  • CXCR2
  • chemokines
  • squamous cell carcinoma

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