Giulia Bertolini

The identification of lung tumor-initiating cells and associated markers may be useful for optimization of therapeutic approaches and for predictive and prognostic information in lung cancer patients. CD133, a surface glycoprotein linked to organ-specific stem cells, was described as a marker of cancer-initiating cells in different tumor types. Here, we report that a CD133+, epithelial-specific antigen-positive (CD133+ESA+) population is increased in primary nonsmall cell lung cancer (NSCLC) compared with normal lung tissue and has higher tumorigenic potential in SCID mice and expression of genes involved in stemness, adhesion, motility, and drug efflux than the CD133(-) counterpart. Cisplatin treatment of lung cancer cells in vitro resulted in enrichment of CD133+ fraction both after acute cytotoxic exposure and in cells with stable cisplatin-resistant phenotype. Subpopulations of CD133+ABCG2+ and CD133+CXCR4+ cells were spared by in vivo cisplatin treatment of lung tumor xenografts established from primary tumors. A tendency toward shorter progression-free survival was observed in CD133+ NSCLC patients treated with platinum-containing regimens. Our results indicate that chemoresistant populations with highly tumorigenic and stem-like features are present in lung tumors. The molecular features of these cells may provide the rationale for more specific therapeutic targeting and the definition of predictive factors in clinical management of this lethal disease.

Several lines of compelling pre-clinical evidence identify chemotherapy as a potentially double-edged sword: therapeutic efficacy on the primary tumor may in fact be counterbalanced by the induction of tumor/host reactive responses supportive for survival and dissemination of cancer cell subpopulations. This paradoxical effect of chemotherapy can affect different districts such as the primary tumor, the circulation and distant organs by simultaneously shaping properties and composition of tumor and stromal cells. At the primary tumor site, chemotherapy has been reported to promote selection of chemoresistant and disseminating tumor cells endowed with properties of cancer stem cells (CSCs) through activation of autocrine and paracrine self-renewing/survival pathways promoted jointly by therapy-selected tumor and stromal cells. Resistant CSCs represent seeds for tumor relapse and increased infiltration by immune cells, together with enhanced vascular permeability induced by chemotherapy, facilitates tumor cells intravasation, the first step of the metastatic cascade. As a consequence of primary tumor/metastasis re-shaping induced by chemotherapy, circulating tumor cells (CTCs) detected during therapy can display a shift towards a more mesenchymal and stem-like phenotype, conductive to increased ability to survive in the circulation and seed distant organs. At the metastatic site, host responses to therapy activate inflammatory pathways that ultimately facilitate tumor cells extravasation and metastatic colonization. Finally, cooperation of immune cells and endothelial cells at perivascular niches favors the extravasation of tumor cells endowed with high potential for metastasis initiation and protects them from chemotherapy. This review highlights the paradoxical pro-metastatic effects of chemotherapy linking reactive responses to treatment to tumor relapse and metastasis formation through primary tumor remodeling and generation of a favorable pro-metastatic niche.

Cancer cells within a tumor are functionally heterogeneous and specific subpopulations, defined as cancer initiating cells (CICs), are endowed with higher tumor forming potential. The CIC state, however, is not hierarchically stable and conversion of non-CICs to CICs under microenvironment signals might represent a determinant of tumor aggressiveness. How plasticity is regulated at the cellular level is however poorly understood. To identify determinants of plasticity in lung cancer we exposed eight different cell lines to TGFβ1 to induce EMT and stimulate modulation of CD133(+) CICs. We show that response to TGFβ1 treatment is heterogeneous with some cells readily switching to stem cell state (1.5-2 fold CICs increase) and others being unresponsive to stimulation. This response is unrelated to original CICs content or extent of EMT engagement but is tightly dependent on balance between epithelial and mesenchymal features as measured by the ratio of expression of CDH1 (E-cadherin) to SNAI2. Epigenetic modulation of this balance can restore sensitivity of unresponsive models to microenvironmental stimuli, including those elicited by cancer-associated fibroblasts both in vitro and in vivo. In particular, tumors with increased prevalence of cells with features of partial EMT (hybrid epithelial/mesenchymal phenotype) are endowed with the highest plasticity and specific patterns of expression of SNAI2 and CDH1 markers identify a subset of tumors with worse prognosis. In conclusion, here we describe a connection between a hybrid epithelial/mesenchymal phenotype and conversion to stem-cell state in response to external stimuli. These findings have implications for current endeavors to identify tumors with increased plasticity.

Metastasis is the main reason for lung cancer-related mortality, but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here, we show that CD133(+)/CXCR4(+) cancer-initiating cells (CIC) directly isolated from patient-derived xenografts (PDX) of non-small cell lung cancer are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133(+)/CXCR4(+) cells in residual tumors and their metastatization. Immunophenotypic analysis of lung tumor cells intravenously injected or spontaneously disseminated to murine lungs demonstrated the survival advantage and increased colonization ability of a specific subset of CD133(+)/CXCR4(+) with reduced expression of epithelial cell adhesion molecule (EpCAM(-)), which also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cells from lungs of PDX-bearing mice enriched for CD133(+)/CXCR4(+)/EpCAM(-) CICs are highly tumorigenic and metastatic. Importantly, microenvironment stimuli eliciting epithelial-to-mesenchymal transition, including signals from cancer-associated fibroblasts, are able to increase the dissemination potential of lung cancer cells through the generation of the CD133(+)/CXCR4(+)/EpCAM(-) subset. These findings also have correlates in patient samples where disseminating CICs are enriched in metastatic lymph nodes (20-fold, P = 0.006) and their detection in primary tumors is correlated with poor clinical outcome (disease-free survival: P = 0.03; overall survival: P = 0.05). Overall, these results highlight the importance of specific cellular subsets in the metastatic process, the need for in-depth characterization of disseminating tumor cells, and the potential of therapeutic strategies targeting both primary tumor and tumor-microenvironment interactions.