Direct, in vivo
interrogation of the effects of components of the tumor microenvironment on gene expression offers significant advantages over similar in vitro
approaches. First, EF5-LCM-MGEP offers the opportunity to analyze the effects of one stress (in this case low oxygen tension) in a physiological setting which invariably includes additional parameters such as nutrient availability, low pH and high interstitial pressure, all of which are known to influence gene expression in tumor cells (38
). Second, as our results indicate, this type of analysis has the potential to uncover stress responses that might be unanticipated or simply could not be interrogated under in vitro
conditions. In our study, we observed a pronounced downregulation of a large number of mRNAs associated with immune system regulation and function in hypoxic areas, raising the intriguing possibility that hypoxic tumor areas represent an immunoprivileged tumor niche. If confirmed in other types of tumors, this finding would predict a new resistance factor for hypoxic tumors.
This in vivo
approach of gene expression analysis is not without some limitations. For example, without further analysis, it cannot be determined whether the observed decreases in gene expression of immune response gene products under hypoxia is the result of direct downregulation of transcriptional programs or the consequence of over/under-representation of one type of lymphocyte or macrophage over another in hypoxic regions. Tumor-Associated Macrophages (TAMs) have been shown to segregate into two broadly-defined classes: The pro-oxidant, cytotoxic and anti-tumorigenic type, M1 and the pro-angiogenic pro-tumorigenic type M2 (40
). Our findings indicate that expression of genes associated with the M1 phenotype (Ly6C, Cxcl9, Cxcl10
) are significantly under-represented in hypoxic vs. normoxic areas. This could arise either from (a) an active repression of M1-associated genes under hypoxic stress, or (b) exclusion of M1 or attraction of M2 in hypoxic compared to normoxic tumor areas. Our findings that hypoxic areas are largely devoid of CD8+
cells (cytotoxic T lymphocytes) argue for the latter mechanism. However, it is formally possible that CD8 expression may also be downregulated by hypoxia, even though it did not appear in our list of downregulated mRNAs. Intriguingly, in a recently published study, Doedens et al
. reported that TAMs under hypoxic conditions negatively regulate T cell function through HIF-1α (44
). Although in this study the localization of T cells in solid tumors with relation to hypoxic areas was not analyzed, these results suggest that hypoxia may have additional repressive effects on T-cell activity, thereby promoting tumor aggressiveness. Additional studies with specific markers and immunohistochemical localization of the two types of macrophages and T cells with respect to hypoxic regions in vivo
would help elucidate the precise mechanism and these studies are currently under way in our laboratory.
Our analysis has revealed a significantly higher number of mRNAs affected by hypoxia in vivo
compared to in vitro
. The increased number of upregulated mRNAs in vivo
vs. in vitro
is in agreement with other studies demonstrating a more robust gene upregulation in response to stresses in vivo
. For example, Khodarev et al
., demonstrated that ionizing radiation induced significantly higher up-regulation of genes in xenografts than in in vitro
) and Camphausen et al
demonstrated that the tumor microenvironment can exert substantial influence on gene expression in gliomas in response to ionizing radiation (46
). While several mechanisms could underlie this difference for hypoxia, it is noteworthy that there appears to be a trend towards higher number of upregulated mRNAs from 6h hypoxia to 16h hypoxia (from 37 to 66, respectively). It is possible that in vivo
, where moderate hypoxic conditions can persist for more than 16h without significantly affecting cell survival, the prolonged exposure to low oxygen can result in a higher number of upregulated genes.
Identification of an mRNA “hypoxic signature” in solid tumors has important implications with clinical significance. For example, several proteins such as GLUT-1, VEGF, Osteopontin, etc. have been shown to correlate with, and predict a poorer patient prognosis (47
). More recently, hypoxia-induced miRNAs such as mir210 have been associated with increased metastasis and poorer patient outcome (48
). These studies are almost invariably initiated by in vitro
analyses of gene expression patterns and subsequent immunohistochemical analysis (in the case of proteins) or PCR analysis (in the case of miRNA) in patients’ samples. The EF5-LCM-MGEP method offers a more systematic approach in which multiple targets are identified and validated in vivo
and then a select few targets of interest are moved to the clinic. Our approach is readily amenable to further analysis of gene expression to include miRNA and protein expression using miRNA microarrays and proteomics respectively. Preliminary studies in our laboratory have demonstrated feasibility for the analysis of miRNAs using a similar approach and we are in the process of obtaining the hypoxic miRNA expression profile in human solid tumors. It is therefore conceivable that in the near future, a systems-level analysis of the hypoxic response in solid tumors will be feasible which could uncover novel targets for therapeutic intervention.