The heterogeneity of cancer cells introduces significant challenges in designing effective therapeutic approaches. This tremendous flexibility is maintained via the expression of multiple isoforms mainly produced by alternative splicing, in a non-uniform distribution, constituting probably one cause of treatment failure. Characterizing isoforms heterogeneity might therefore allow for a better understanding of tumorigenesis and facilitate the development of more suitable therapeutic strategies. In this study we have explored the level of tumor heterogeneity by studying HLA-G as paradigm in clear cell renal cell carcinoma (ccRCC).

HLA-G is a non-classical HLA-class I molecule whose best known role is to inhibit the cytotoxic activity of natural killer and T-cells. This molecule was first described to play a crucial role in feto-maternal tolerance. HLA-G expression is not ubiquitous, in contrast to other HLA-class I molecules. Its physiological expression is mainly restricted to extravillous cytotrophoblasts. However, under pathological conditions, HLA-G expression can be induced in almost all tissues. Particularly, HLA-G has been found in most of the tumors analyzed, such as ccRCC, the most common human renal malignancy, in which a high incidence of HLA-G was described. More recently, we have reported novel HLA-G isoforms in addition to the seven already reported that include spliced forms having an extended 5’-region and lacking the transmembrane and/or alpha1 domains. In the present study we have extended our analysis by simultaneous assessment of immunohistochemistry (IHC) labeling and RT-PCR. Combining both methods was necessary since the specificity of the different antibodies have been questioned. The study of tumors retrieved from ccRCC patients reveal that the expression of HLA-G is highly variable among tumors and distinct areas of the same tumor. In particular, we have repeatedly noticed that ccRCC samples contain eosinophilic hyaline globules exhibiting glassy appearance with a clear surrounding halo. These hyaline globules represent a well-characterized morphologic and functional entity associated with a more aggressive behavior and seem to be directly linked to autophagy defects. Interestingly, we observed that hyaline globules are associated with enhanced expression of HLA-G. Moreover, the novel isoforms were also detected in placental trophoblast cells demonstrating that they did not arise from spontaneous mutations in the process of tumor formation. The absence of alpha1 domain from one of the membrane-bound isoforms prevents its detection by immunostaining since all antibodies recognize an epitope located into the alpha1 domain common to all HLA-G isoforms. Therefore, we also report here the development of a lentivirus-vector cellular model to specifically analyze the characteristics of this particular novel isoform. Collectively, our approach extends the portrait of HLA-G isoforms expression and provides data that might facilitate the development of more-effective personalized therapies.