Regulated Expression of CCL21 in the Prostate Tumor Microenvironment Inhibits Tumor Growth and Metastasis in an Orthotopic Model of Prostate Cancer

TRAMPC2 tumors grow progressively in immune competent mice suggesting that these cells induce a weak or inefficient anti-tumor immune response. This may reflect the ability of the TRAMPC2 TME to impair DC (CD11c+ cells) function. CD11c has been used here to identify DCs, although it can also be expressed by activated T and B cells as well as natural killer (NK) cells. However, intratumoral T cells remain quiescent in the TRAMP TME because they do not express the activation antigens CD25 or CD69 (data not shown). Furthermore, T and B cells are not a major infiltrating cell types in TRAMP tumors. NK cells are typically not detected in TRAMP TILs or are present as a trace population and therefore do not contribute significantly to CD11c expression in the TRAMP TME. We observed that the majority of DCs infiltrating TRAMPC2 tumors failed to express normal levels of class II antigens (IA^b), B7.2 and CD40 molecules compared to their counterparts isolated from either normal or tumor bearing spleens (Fig. 1-b). Most of the infiltrating DCs appeared to be myeloid in origin because they did not express CD8α (B-g, h and i and C). Class I antigen (H2D^b) expression was not suppressed by the TME as equivalent levels of expression were observed on intratumoral and splenic DCs (Fig. 1-b; g, h and i). Surprisingly, CD86 expression, but not CD80, was suppressed suggesting differential regulation of B7 family members within the prostate TME (Fig. 1-c). As expected, expression of the chemokine receptor CCR7 was down-regulated relative to normal spleen (Fig. 1-c). In contrast, DC expression of PDL2 shown to inhibit the activation and cytokine production of CD4⁺ T cells [16] was elevated on intratumoral DCs relative to normal splenic DCs (Fig. 1-c). Thus, these data suggest that tumor-associated DCs are immature because they fail to express a number of cell surface markers associated with DC maturation.

Previous studies showed that the presence of CCL21 in tumors promotes the infiltration of DCs and T cells that enhanced the anti-tumor immune response and inhibited tumor growth [6, 17]. We examined whether direct intratumoral expression of CCL21 via gene-modified TRAMPC2 cells would inhibit tumor growth and metastatic disease in this model. We therefore transfected TRAMPC2 cells with both the repressor and CCL21 tet-inducible expression vectors. Six antibiotic resistant TRAMPC2/TR/CCL21 clones were isolated that possessed low constitutive expression of the chemokine and 12-to 60-fold induction of CCL21 in the presence of tetracycline. Three out of 6 lines maintained the tet-inducible expression of CCL21 (termed TRAMPC2/TR/CCL21) after 3 and 8 additional passages although clone 6 had lower levels of inducible expression after 8 passages (Fig. 2-a). To establish a cell line that grows and maintains regulated expression of CCL21 in vivo, TRAMPC2/TR/CCL21 tumor cells (Fig. 2a, clone 4) were implanted into the prostate gland of nine mice. One mouse died without evidence of a palpable prostate tumor. Six mice developed palpable tumors that were excised and clonal outgrowths were obtained without selection antibiotics. Outgrowths from two tumors (M5, M6) were no longer tet-inducible and were not further studied (Table 1). Seventy clonal lines were obtained from the remaining four tumors of which ten were inducible for CCL21 expression (Fig. 2b and Table 1). Clonal outgrowths derived from mouse 1 (M1) generally had low constitutive CCL21 levels with relatively weak induction for CCL21. The remaining clones demonstrated higher tet-induced CCL21 secretion but were “leaky” (high constitutive levels). Because the clonal outgrowths from intraprostatic tumors were isolated and grown in the absence of selection media, the relatively modest induction of CCL21 production may indicate that TRAMPC2/TR/CCL21 tumor cells lost or silenced the CCL21 gene during in vivo growth. To test this hypothesis and to enrich for tumor cells with stable tet-inducible expression of CCL21, the 8 weakly inducible clonal lines from mouse 1 (M1.1-1.19) and 4 (M4.2, M4.4) were pooled to generate TRAMPC2/TR/CCL21-L1. The remaining two lines were also pooled to produce TRAMPC2/TR/CCL21-L2. Both populations were then subjected to antibiotic selection. Fig. 2c demonstrates that growth in selection media enhanced CCL21 production by TRAMPC2/TR/CCL21-L1 cells (Line 1) approximately 5-fold without increasing constitutive CCL21 levels. Although TRAMPC2/TR/CCL21-L2 (Line 2) cells had higher background expression relative to Line 1, tetracycline induced much higher levels of CCL21 production. These data indicate that both these lines have the capacity to grow both in vitro and in vivo following orthotopic implantation.

TRAMPC2/TR/CCL21 clones (L1 and L2) displayed high levels of tet-inducible expression of CCL21 and grew in vivo. We next wanted to test whether CCL21 expression in the TME enhances survival of mice implanted orthotopically with prostate tumor cells. Therefore, mice were randomly divided into two groups and one group received doxycycline in their drinking water starting the day after surgical implantation of TRAMPC2/TR/CCL21-L2. Mice were sacrificed and tumors explanted for in vitro growth when mice showed signs of morbidity. As demonstrated in Fig. 3a (left panel), prolonged survival was observed in doxycycline-treated mice. Control mice all died by day 32, whereas, doxycycline treated mice lived up to 50 days post implantation. Low levels of CCL21 were detected in the serum of 25% of tumor bearing mice treated with doxycycline and was not detected in the serum of control mice. Analysis of clonal lines derived these tumors demonstrated that <10% were capable of inducible expression of CCL21 (right panel). All these cell lines eventually lost inducible expression after a few in vitro passages (data not shown). Thus, tumor growth in vivo was associated with loss of inducible expression of CCL21. This may have contributed to the limited growth inhibition and eventual outgrowth of primary prostate tumors observed in these mice.

To determine whether prolonged survival induced by intratumoral CCL21 expression reflected inhibition of primary tumor growth and/or inhibition of metastatic disease, we implanted TRAMPC2/TR/CCL21-L2 orthotopically into the total of 18 animals in two separate experiments (8 and 10 mice, respectively). In each experiment the mice were divided into two groups with one group receiving doxycycline in their drinking water one day after tumor implantation. Mice were sacrificed when moribund and tumors, draining lymph nodes, lungs and pancreases removed for measurements and assessment of metastatic disease. One of the mice given doxycycline in the first experiment and two from the control group in the second experiment died shortly after tumor implantation and therefore were excluded from this analysis. Tumors grew in all mice irrespective of whether they received doxycycline in their drinking water. However, Fig. 3b demonstrates that tumors excised from doxycycline-treated mice weighed less (left panel) and were smaller in size (right panel) than tumors excised from control animals. As expected, all control mice had metastases in draining periaortic lymph nodes as well as metastases in their lung in the majority of mice. A smaller subset also had disseminated disease to the pancreas (panel C). In contrast, treated mice had reduced frequency of metastasis to lymph nodes and lungs with no metastases to the pancreas. These data suggest that even limited and transient expression of CCL21 in TRAMP TME suppresses primary tumor growth as well as metastatic disease to draining lymph nodes and distant organs.

The data presented above demonstrated that the vast majority of TRAMPC2/TR/CCL21 tumor cells no longer displayed inducible CCL21 induction following orthotopic implantation. Two possibilities mechanisms were next considered to explain this observation: loss of the transgene or alternatively, silencing of the promoter. To test the first possibility DNA was extracted from TRAMPC2/TR/CCL21-L2 tumor pieces and cloned lines isolated and expanded to generate sufficient DNA for PCR analysis using specific primers to amplify the transfected CCL21 gene. It is apparent from Fig. 4 (panel A) that outgrowths obtained from orthotopic TRAMPC2/TR/CCL21 tumors still contained the CCL21 transgene. The absence of a product in the control mouse DNA confirmed that the primers did not amplify endogenous CCL21 gene (lane 9). To test the possibility that the promoter was silenced by methylation, we evaluated the methylation pattern of the CMV promoter. DNA isolated from tumor pieces or clonal lines were bisulfite treated and PCR reactions were performed using primers complementary to a region of CMV promoter not containing methylation sites (oligos 1) or a pair of primers complementary to a region of CMV promoter which contains methylation sites (oligos 2). If the promoter is not methylated, a PCR product forms with both primers, whereas, a single product is only detected with oligos 1 if the promoter is methylated [18]. Fig. 4b demonstrates that when the pcDNA4/TO/CCL21 plasmid was tested following bisulfite conversion, PCR reactions with both primers produced a product indicating that the original plasmid DNA was not methylated. In contrast, when DNA was extracted from two excised TRAMPC2/CCL21-L2 tumors (M1 and M2, Fig. 3a), both promoters appeared to be methylated, however, when a clonal outgrowth derived from tumor M1 was tested, PCR products formed with both primers suggesting that the section of tumor excised for clonal expansion had a functional promoter (not methylated). These data indicate that during tumor growth in the prostate gland, the promoter is variably methylated. Thus, in some sections of the tumor, the promoter may still be functional. This may explain why we detected some low-grade induction of CCL21 in some clonal lines derived from explants of TRAMPC2/TR/CCL21-L2 tumors (Fig. 3a, right panel).